Connexins are transmembrane proteins that create intercellular aqueous channels connecting the cytoplasms of adjacent cells. Gap junctions permit the passive transfer of small molecules directly from cell to cell and play a critical role in the function of normal organs, such as the depolarizing current in the heart, electrotonic conduction in nerves, metabolic homeostasis in the lens and ear, and embryonic development. Disruption of gap junction function by mutations in connexin genes causes several human diseases, including peripheral neuropathy (CMTX), deafness, cataract formation, skin diseases, and may contribute to the development and maintenance of cancer. Understanding the mechanisms that regulate the function of these unique intercellular channels is an important goal. This proposal will continue our investigation of the regulation of connexin43 (Cx43) channels by phosphorylation induced by the Src tyrosine kinase oncoprotein and begin the exploration of another regulatory mechanism involving the interaction of novel proteins with Cx43. Specific Aim 1 will investigate the involvement of the P-Y247 site and other domains of Cx43 in the Src-induced disruption of Cx43 gap junction channels. Specific Aim 2 will investigate the interaction between Cx43 and CIP75 and the role CIP75 may play in the proteasomal degradation of Cx43 in mammalian cells. Specific Aim 3 will investigate the interaction between Cx43 and CIP85 and the possible role of CIP85 in the vesicular trafficking and degradation of Cx43. The novel CIP75 and CIP85 proteins contain multiple interaction domains that may contribute to the modulation of Cx43 levels and function. These aims will be accomplished by: in vitro and cellular protein-protein interaction studies, subcellular localization of proteins in fixed and live cells by laser confocal microscopy, analysis of site-specific or deletion mutants, inhibition of protein function by siRNA, dominant negative inhibitors, and the analysis of exogenous wild-type and mutant proteins introduced into cells by DMA transfection, retroviral infection, or direct protein transduction.